It is desirable for the biotech industry to provide microbial strains devoid of antibiotic resistance markers comprising several chromosomally integrated copies of a gene of interest, for the industrial high yield production of polypeptides. Antibiotic marker genes have traditionally been used as a means to select for strains carrying multiple copies of both the marker gene and an accompanying expression cassette coding for a polypeptide of industrial interest. Amplification of the expression cassette by increasing the copy number in a microbiological production strain was desirable because there is very often a direct correlation between the number of copies and the final product yields. Amplification methods using antibiotic selection markers have been used extensively in many host strains over the past 20 years and have proven to be a very efficient way to develop high yielding production strains in a relatively short time, irrespective of the expression level of the individual expression cassettes.
It has previously been shown in Bacillus that a galactose epimerase-encoding gene expressed from a crippled low-level promoter could be used as a selection marker for site-specific genomic integration of tandemly amplified copies of a product-gene (WO 2001/90393; Novozymes A/S). However, no similar systems have been described for fungal host cells.
A riboswitch is part of an mRNA molecule that can bind directly to a small target molecule without a protein being involved. Binding of the small target molecule will affect the translation of the mRNA [Nudler E, Mironov A S (2004). “The riboswitch control of bacterial metabolism”. Trends Biochem Sci 29 (1): 11-7; Vitreschak A G, Rodionov D A, Mironov A A, Gelfand M S (2004). “Riboswitches: the oldest mechanism for the regulation of gene expression?”. Trends Genet 20 (1): 44-50; Tucker B J, Breaker R R (2005). “Riboswitches as versatile gene control elements”. Curr Opin Struct Biol 15 (3): 342-8; Batey R T (2006). “Structures of regulatory elements in mRNAs”. Curr Opin Struct Biol 16 (3): 299-306].
In the filamentous fungal cell Aspergillus oryzae expression of the thiA and nmtA genes are regulated by riboswitches that bind thiamine pyrophosphate (TPP) and controls alternative splicing to conditionally produce an upstream Open Reading Frame (ORF), thereby affecting the expression of the downstream gene(s). The thiA riboswitch of A. oryzae contains a nuclear pre-mRNA intron, a spliceosomal intron, which is involved in facilitating the alternative splicing.
Another filamentous fungal riboswitch has been found in Aspergillus nidulans, where the agaA gene is regulated by mRNA arginine-binding, thereby facilitating alternative splicing [Borsuk, P. et al. (2007). “L-Arginine influences the structure and function of arginase mRNA in Aspergillus nidulans”. Biol Chem. 388: 135-144].
In order to comply with the current demand for recombinant fungal production host strains devoid of antibiotic markers, we have looked for possible alternatives to produce multi-copy host strains.